We are studying the control mechanisms of the expression of the gal operon of E. coli. We have so far shown that the operon is controlled by two promoters, which are modulated by cyclic AMP in opposite ways. Both the promoters are negatively regulated by a gal repressor protein. From our genetic and DNA sequence studies, we had previously proposed that each of the two gal promoters is negatively regulated by two operator elements, one of which (OE) is located upstreams to the promoters and the other (OI) insidse the gaIE structured gene. Using a new polyacrylamide gel electrophoresis method for studying DNA-protein interactions, we have now demonstrated sequence-specific binding of purified gal repressor to both OE+ and OI+ DNA but not to mutant OE(C) and OI(C). By DNase protection experiments we have visualized repressor binding to OE at -50 to -73 positions and to OI at +45 to +68 positions of the gal DNA. This confirms the genetically assigned operator role of OE and OI. We have also shown that repressor does not compete with cAMP.CRP or RNA Polymerase to bind to gal DNA, suggesting steric hindrance as an unlikely mechanism for gal repression. We have also determined the complete DNA sequence of the entire gal operon by Dideoxy method of Sanger.